The aim of this research is to characterize the chemical steps involved in the translocation of short chain fatty acids, including ketone bodies, into bacterial cells and mitochondria. The majority of our ongoing work centers on an acetyl CoA:acetoacetate CoA transferase from Escherichia coli which catalyzes the group translocation of acetoacetate and other short chain fatty acids into these bacterial cells. The enzyme has been well characterized in our laboratory and our proposed research deals with a further investigation of the structure and catalytic mechanism of the enzyme. These studies will employ immunochemical methods as well as covalent modification of the enzyme. We are also investigating two distinct conformational changes that occur in the catalytic pathway--one resulting from binding acyl CoA substrate and a second resulting from formation of a covalent enzyme-CoA intermediate. We have proposed to investigate the transport of D(-)-3-hydroxybutyrate into heart mitochondria. 3-Hydroxybutyrate is a preferred respiratory substrate for heart mitochondria. The kinetic constants of the transport system will be compared with the kinetic constants of subsequent enzymatic steps in order to ascertain how substrate and coenzyme availability may regulate the oxidation of 3-hydroxybutyrate to acetyl CoA.